1. Field of the Invention
The present invention relates to a novel CD33-like protein. In particular, isolated nucleic acid molecules are provided encoding the CD33-like protein. Recombinant CD33-like polypeptides are also provided as are recombinant vectors and host cells. The invention further provides methods useful during tumor or inflammatory disease diagnosis or prognosis and therapeutic treatments targeting cells expressing CD33-like polypeptides.
2. Background Information
CD33 was originally defined on human myeloid cells by a panel of monoclonal antibodies (MoAbs) that recognize a glycoprotein of 67 kD that is restricted in its expression to cells of the hematopoietic system (Peiper S. C. et al., in Knapp W, Dorken B, Gilks W R, Rieber E P, Schmidt R E, Stein H, von dem Borne A E G (eds): Leucocyte Typing IV White Cell Differentiation Antigens. Oxford, UK, Oxford University 1989, p 814; Pierelli L. et al., Br J Haematol 84:24 (1993); Andrews R. G. et al., Blood 62:124, (1983); Griffin J. D., et al., Leuk Res 8:521 (1984)). CD33 is absent from hematopoietic stem cells but is first detected on a subpopulation of mixed colony-forming cells (Pierelli L. et al., Br J Haematol 84:24 (1993); Griffin J. D. et al., Leuk Res 8:521 (1984)). Expression then continues along the myelomonocytic pathway until it is downregulated on granulocytes but retained by monocytes and tissue macrophages. (Pierelli L. et al., Br J Haematol 84:24 (1993); Bernstein I. D. et al., J Clin Invest 79:1153 (1987)). The expression pattern of CD33 within the hematopoietic system indicates a potential role in the regulation of myeloid cell differentiation. However, despite its initial identification over 10 years ago (Andrews R. G. et al, Blood 62:124 (1983)), the functions and binding properties of CD33 have remained obscure.
CD33 MoAbs are of great importance in the immunodiagnosis of acute leukemias, allowing distinction between myeloid leukemic cells (acute myeloid leukemia (AML) French-American-British classification MI-7) and the usually CD33-negative cells of lymphoid origin. (Griffin J. D. et al., Leuk Res 8:521 (1984); Matutes E. et al., Haematol Oncol 3:179 (1985); Bain B.J.: Immunological cytogenetics and other markers, in Bain B J (ed): Leukaemia Diagnosis: A Guide to FAB Classification. London, UK, Gower Medical, 1990, p 61). This is especially valuable for the more immature forms of AML, where morphologic criteria are insufficient yet correct categorization is essential for prognostic predictions and the choice of therapy. CD33 MoAbs have also been used in preliminary therapeutic trials, principally for purging of the bone marrow of AML patients, either before transplantation or in case of diseases that are resistant to chemotherapy. (Robertson M. J. et al., Blood 79:2229 (1992); Applebaum F. R. et al., Transplantation 54: 829 (1992); Caron P. C. et al., Cancer 73:1049 (1994)). Thus, due to the importance of CD33, there is a clear need to identify and isolate nucleic acid molecules encoding additional polypeptides having CD33-like protein activity.
The present invention provides isolated nucleic acid molecules comprising a nucleic acid sequence encoding a CD33-like protein whose amino acid sequence is shown in FIGS. 1A-1C (SEQ ID NO:2) or a fragment of the polypeptide. The CD33-like protein gene contains an open reading frame encoding a protein of about 551 amino acid residues whose initiation codon is at position 37-39 of the nucleotide sequence shown in FIGS. 1A-1C (SEQ ID NO:1), with a leader sequence of about 15 amino acid residues, and a deduced molecular weight of about 60 kDa. The amino acid sequence of the mature CD33-like protein is shown in FIGS. 1A-1C (amino acid residues from about 1 to about 536 in SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the CD33-like protein having the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the CD33-like protein having the complete amino acid sequence in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) a nucleotide sequence encoding the mature CD33-like protein having the amino acid sequence at positions from about 1 to about 536 in SEQ ID NO:2; (d) a nucleotide sequence encoding the CD33-like protein having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97521; (e) a nucleotide sequence encoding the mature CD33-like protein having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97521; (f) a nucleotide sequence encoding the CD33-like protein extracellular domain; (g) a nucleotide sequence encoding the CD33-like protein transmembrane domain; (h) a nucleotide sequence encoding the CD33-like protein intracellular domain; (i) a nucleotide sequence encoding the CD33-like protein intracellular and extracellular domains with all or part of the transmembrane domain deleted; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a CD33-like protein having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of CD33-like polypeptides or fragments thereof by recombinant techniques.
The polypeptides of the present invention include the polypeptide encoded by the deposited cDNA, the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide), as well as polypeptides having an amino acid sequence at least 95% identical, more preferably, at least 96% or 99% identical, to the amino acid sequence of the polypeptide encoded by the deposited cDNA or the polypeptide of SEQ ID NO:2.
The invention further provides an isolated CD33-like protein having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the CD33-like protein having the complete 551 amino acid sequence, including the leader sequence shown in SEQ ID NO:2; (b) the amino acid sequence of the CD33-like protein having the complete 551 amino acid sequence, including the leader sequence shown in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) the amino acid sequence of the mature CD33-like protein (without the leader) having the amino acid sequence at positions from about 1 to about 536 in SEQ ID NO:2; (d) the amino acid sequence of the CD33-like protein having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 97521; (e) the amino acid sequence of the mature CD33-like protein having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97521; (f) the amino acid sequence of the CD33-like protein extracellular domain; (g) the amino acid sequence of the CD33-like protein transmembrane domain; (h) the amino acid sequence of the CD33-like protein intracellular domain; and (i) the amino acid sequence of the CD33-like protein intracellular and extracellular domains with all or part of the transmembrane domain deleted.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a CD33-like polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a CD33-like polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a CD33-like polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above. Such antibodies are useful diagnostically or therapeutically as described below.
The invention further provides a method useful during tumor or inflammatory disease diagnosis, which involves assaying the expression level of the gene encoding the CD33-like protein or the gene copy number in mammalian cells or body fluid and comparing the gene expression level or gene copy number with a standard CD33-like protein gene expression level or gene copy number, whereby an increase in the gene expression level or gene copy number over the standard is indicative of certain tumors or inflammatory disease. By the invention, the above-described method is further useful as a prognostic indicator.
In another embodiment, an in vitro method is provided for purging leukemic hematopoietic cells from the autografts of patients with leukemia. The method involves removing CD33-like antigen-containing hematopoietic cells from bone marrow obtained from the patient with an anti-CD33-like protein monoclonal antibody (MoAb) and complement.
In a further embodiment, the invention provides an in vivo method for selectively killing or inhibiting growth of tumor cells expressing the CD33-like antigen of the present invention, which involves administering to a patient an effective amount of an antagonist to inhibit the CD33-like protein receptor signaling pathway. By the invention, administering such antagonists of the CD33-like protein to a patient is also useful for treating inflammatory disease.
In a still further embodiment, immunotoxins specific for cells expressing the CD33-like protein are provided for selective killing of tumor cells. The immunotoxins of the present invention are further useful according to the method described above for purging leukemic hematopoietic CD33+ cells in vitro.